Phosphorylase kinase is a central enzyme in the regulation of glycogenolysis and is the site of integration of hormonal control. It is a protein of complex overall structure (alpha-beta-gamma-delta)4, and is regulated multiply by both Ca2+ and phosphorylation. In continuation of our past progress, our overall goals for the next grant period are to continue to elaborate the transcriptional regulation of the subunits, to evaluate the route of subunit assembly into holoenzyme, and to further define the functional domains of each subunit and the interactions between them. We will approach these questions by a combination of both in vivo and in vitro methods. We have shown in the bupivacaine-treated EDL (extensor digitorum longus) skeletal muscle that there is a coordination of transcription of the alpha, beta and gamma subunits during subsequent muscle regeneration. We will use this system to study subunit synthesis and holoenzyme assembly, and extend upon it to evaluate the potential neural regulation of phosphorylase kinase formation. Coupled with these studies will be a continuation of our investigations on the structure of the genes for each of the subunits, and the modes for transcriptional regulation. We also propose to further our investigations on the in vitro expression of phosphorylase kinase subunits, with the goal of assessing the functional domains within each subunit and their modes of interaction. One of our first targets will be the gamma subunit, examining its interactions with protein substrates, calmodulin and the other subunits. Also, starting with the gamma-delta complex, we will examine the topographical relationship between subunits. An eventual aim is to achieve the in vitro assembly of the holoenzyme.